C. elegans spermatozoa lacking spe-45 are incapable of fusing with the oocyte plasma membrane

C. elegans spe-9 class genes encode sperm proteins with indispensable roles during fertilization. We have previously reported that spe-45 belongs to the spe-9 class, based on the finding that self-sperm of spe-45(tm3715) hermaphrodites were not consumed by fertilization. In this study, we directly observed live fertilization in the spermatheca of fem-1(hc17) females after mating with spe-45(tm3715) males. As expected, it was clearly shown that spe-45 mutant spermatozoa failed to fuse with the oocyte plasma membrane. Thus, our live imaging system for C. elegans fertilization seems to be useful for evaluation of the functions of male and female gametes.

spe-45(tm3715) spermatozoa fail to fuse with oocytes in the spermatheca of fem-1(hc17) females: (A) Gross structures of mouse IZUMO1 and SPACA6 and C. elegans SPE-45. All the proteins shown here are sperm transmembrane (TM) proteins with a single immunoglobulin (Ig)-like domain that are essentially required for gamete fusion. The domain architectures of each protein were predicted by the SMART program (http://smart.embl-heidelberg.de). AA, amino acid; N, the amino-terminus; C, the carboxy-terminus; +, positively charged region; and -, negatively charged region. Numbers of "+" and "-" symbols represent relative numbers of basic and acidic amino acids, respectively. This panel was prepared based on the previous reports (Singaravelu et al., 2015;Nishimura et al., 2015;Nishimura and L'Hernault, 2016). (B) Time-lapse analysis on the number of self-sperm in the spermatheca of spe-45 and other spe-9 class mutant hermaphrodites. Data shown in this table are based on the assumption that the number of self-sperm in each spermatheca of N2 (wild type, WT), spe-9(eb19), spe-42(tm2421), and spe-45(tm3715) worms at 24 h post the fourth larval stage (L4) is 100%. This table was prepared according to the previously reported data (Nishimura et al., 2015). (C) Live imaging of fertilization between WT or spe-45 mutant spermatozoa and WT oocytes. We generated oxIs318; him-5(e1490) and oxIs318; spe-45(tm3715); him-5(e1490) males as WT and spe-45 mutant, respectively, and these two strains produce spermatozoa of which nuclei are fluorescently labeled with mCherry (magenta). We also raised fem-1(hc17); ltIs38 hermaphrodites at 25°C as WT females, in which the oocyte plasma membrane (PM) carries a green fluorescent protein (GFP, green). After mating of the females with WT (left) or spe-45 mutant (right) males, live fertilization occurring in the spermatheca was observed by two-color 3D time-lapse spinning-disc confocal microscopy, as previously reported (Takayama and Onami, 2016). Still images were then acquired from movies recording the live fertilization. The white arrowhead indicates a fluorescence gap, which presumably shows that sperm-oocyte fusion occurs at the site. While the fluorescence gap region constantly appeared on the oocyte PM during fertilization by WT spermatozoa (n = 8), spe-45 mutant spermatozoa could not yield the arc-shaped GFP signals on the oocyte surface (n = 8). Scale bar, 20 µm.

Description
Gamete fusion is a pivotal step during fertilization to create an organism of the next generation. In C. elegans, since oocytes have no thick egg coat like the zona pellucida, perhaps spermatozoa directly bind to and fuse with the oocyte plasma membrane (PM). Thus, C. elegans is an excellent model to investigate how a spermatozoon and an oocyte fuse together.
Among the male germline functions, spermatogenesis and spermiogenesis are not impaired in spe-45(tm3715) worms (Singaravelu et al., 2015;Nishimura et al., 2015). As shown in Figure 1B, while self-sperm mostly disappeared in the spermatheca of WT hermaphrodites at 72 h post the fourth larval stage (L4), more than half of self-sperm still reside in the spermatheca of spe-9(eb19), spe-42(tm2421), and spe-45(tm3715) hermaphrodites even at the same time point (Nishimura et al., 2015). Thus, all the tested mutants are presumably incapable of completing fertilization (gamete fusion), indicating that spe-45 is a member of the spe-9 class. Additionally, a domain-swapping approach showed that the Ig-like domains of C. elegans SPE-45 and mouse IZUMO1 share a conserved role during fertilization despite only ~9% identity of the entire amino acid sequences of these two proteins (Nishimura et al., 2015).
The data in Figure 1B provide strong but indirect evidence that spe-45 is involved during gamete fusion. On the other hand, we have succeeded in live imaging of fertilization occurring in the spermatheca by two-color 3D time-lapse spinning-disc confocal microscopy (Takayama and Onami, 2016). In this system, we used spermatozoa from males carrying the oxIs318 allele, which produces mCherry-labeled sperm nuclei, and oocytes from ltIs38 hermaphrodites, of which the PM is tagged with a green fluorescent protein (GFP). Therefore, we produced him-5(e1490) and spe-45(tm3715); him-5(e1490) males in the oxIs318 background as WT-like and spe-45 mutant, respectively, and fem-1(hc17) hermaphrodites in the ltIs38 background as WT-like females.
After mating of fem-1 females with WT or spe-45 males, the spermatheca of the females was observed. From movies recording the live fertilization (see Extended Data), we could obtain still images showing a fluorescence gap on the oocyte PM, which was yielded by WT but not spe-45(tm3715) spermatozoa ( Figure 1C). The place indicated by the fluorescence gap region seemed to represent where gamete fusion occurs, based on the previous report (Takayama and Onami, 2016). Therefore, our present findings show direct evidence that spe-45(tm3715) spermatozoa have a defect in sperm-oocyte fusion.
One of the advantages of using C. elegans for reproductive biology is that live fertilization in the spermatheca can be observed through transparent cuticles. Our imaging analysis demonstrates that spermatozoa lacking spe-45 fail to fuse with the oocyte PM, unlike WT spermatozoa. However, it remains to be clarified how each spe-9 class gene acts during gamete fusion. For instance, the data in Figure 1B imply that functional roles of spe-9 and spe-45 might be distinguishable from that of spe-42 during gamete fusion. Our imaging system would provide important clues to solve the point.

Worm maintenance and culture
We maintained and cultured worms at 20ºC unless otherwise stated, according to standard methods (Brenner, 1974). him-5(e1490) was used as WT to produce males. spe-45(tm3715); him-5(e1490) males were obtained by picking GFP-negative males from the SL1491 strain. To generate males from strains without the him-5(e1490) background, we incubated L4 hermaphrodites at 32ºC for 5 h, and the resulting F1 males were picked to use for further experiments. As fem-1(hc17) worms were used as WT females, fertilized eggs of the strain were cultured at 25ºC until they reached the adult stage.

Microscopy
Most worm handling, such as picking and dissecting, were carried out under SZ61 or SZX10 dissecting microscopes (Olympus). To observe GFP signals on the oocyte PM for a screening of the HTN3 strain, we used an Olympus BX53 fluorescent microscope. For imaging experiments, a Nikon Ti-E microscope with the objective lens Plan Apo VC 60xA/1.20 W (Nikon) was used. Confocal microscopy was performed with the spinning-disk confocal unit CSU-X1 (Yokogawa Electric Corp.). The confocal unit was equipped with a solid-state laser lines (488 nm and 561 nm; ALC-501 AOTF, Andor Technology). Time-lapse images were acquired with an EM-CCD camera (iXon DU-897, Andor Technology). Scanning along the z-axis in 3D time-lapse imaging was performed using a piezo stage-positioning system (Nano-Drive, Mad City Labs). Settings for time-lapse imaging were as follows: 18 planes of 0.5 µm z-axis interval, 2 s time interval and 40 ms exposure for each color. The camera and the piezo system were synchronized using a precision controller unit (PCU-100, Andor Technology). Imaging systems were controlled using iQ software (Andor Technology).

Imaging of fertilization in the spermatheca
To obtain females, we raised fem-1(hc17); ltIs38 hermaphrodites at 25ºC. The feminized worms were mated with oxIs318; him-5(e1490) (WT) or oxIs318; spe-45(tm3715); him-5(e1490) (spe-45 mutant) males at 25ºC overnight (female:male = 1:4). Then, the females after mating were immobilized using polystyrene nanoparticles and agarose pads as previously described (Takayama and Onami, 2016). Fertilization occurring in the spermatheca of the immobilized worms was observed by two-color 3D time-lapse spinning-disc confocal microscopy. To generate HTN3, we outcrossed BA17 hermaphrodites to OD58 males at 20ºC. F1 progeny were allowed to produce self-progeny. The F2 offspring were individually separated and allowed to produce F3 self-progeny. We individually picked five animals at the first larval stage (L1) among F3 progeny in each F2 plate, and the F3 larvae were cultured at 25ºC until they developed into adult worms. If all the five F3 worms from the same F2 plate became self-sterile, we judged that the F2 worm homozygously carries the fem-1(hc17) allele. In the F2 fem-1(hc17) worms, we also determined the genotype of ltIs38 by observing GFP-signals on the oocyte PM under an Olympus BX53 fluorescent microscope. A difference of GFP-signal intensities can be distinguishable between heterozygote and homozygote of ltIs38.

Reagents
To generate HTN4, we outcrossed EG4883 hermaphrodites to DR466 males. F1 progeny were allowed to produce selfprogeny. The F2 offspring were individually separated and allowed to produce F3 self-progeny. If a certain F2 plate contains sons as F3 worms, the genotype of oxIs318 in the F2 worm was determined by single-worm PCR (Nishimura et al., 2015), using a primer set of NM3887 and NM3889 (Frøkjaer-Jensen et al., 2008).
To generate HTN5, we outcrossed EG4883 hermaphrodites to SL1491 males. GFP-positive F1 progeny were picked and then allowed to produce self-progeny. GFP-positive F2 offspring were individually separated and allowed to produce F3 self-progeny. If a certain F2 plate contains sons as F3 worms, five GFP-negative F3 males were picked from the plate to examine the genotypes of spe-45 and oxIs318 by single-worm PCR, using primer sets of HN63 and HN64 for spe-45 and NM3887 and NM3888 for oxIs318. If all the five tested F3 worms carried both spe-45(tm3715) and oxIs318 as homozygotes, GFP-positive hermaphrodites in the same F2 plate were maintained as oxIs318; spe-45(tm3715); him-5(e1490); ebEx15-6. As oxIs318; spe-45(tm3715); him-5(e1490) worms were required, GFP-negative animals were picked.
Extended Data: him-5 x fem-1.mov Description: This is a movie file recording live fertilization between WT spermatozoa and WT oocytes.
spe-45; him-5 x fem-1.mov Description: This is a movie file recording live fertilization between spe-45 mutant spermatozoa and WT oocytes.
Extended Data are archived at CaltechData.